1. Field of the Invention
This invention relates to the inhibition of blood coagulation and more particularly to the inhibition of fibrin polymerization by a novel peptide isolated from fibrin Fragment D.sub.1 and to the peptide itself, its isolation, and its additional uses.
2. Description of the Prior Art
Anticoagulants and antithrombic agents are among the most important drugs presently available for the treatment and prevention of intravascular clotting disorders, currently a major cause of death and disease in Western society. The process of thrombus formation is only incompletely understood, but appears to consist of two major stages. Initially, platelets present in the blood aggregate at the site of a blood vessel injury followed by formation of a cross-linked fibrin polymer which binds the forming clot together. Working to counteract this tendency and to dissolve clots are fibrinolytic enzymes that tend to break down the fibrin polymer and dissolve the clot. Therefore, there are at least three steps at which drugs may work: (1) prevention of platelet aggregation, (2) prevention of the formation of the fibrin polymer, and (3) enhancement of fibrinolytic activity. The present invention is related to the second type of action.
One way of providing such an anticoagulant, not fully developed prior to the present invention, is through interference with the polymerization process by use of a competitive inhibitor derived from the natural binding site involved in the polymerization reaction. This could not be realized until the binding site on Fragment D could be located. Although the binding site of polymerization was not specifically located until the present invention, other aspects of the polymerization process had been investigated. Fibrin polymerization in humans begins when fibrinogen is converted to fibrin through a limited proteolytic cleavage by the enzyme thrombin. Thrombin cleaves the bonds A.alpha.16 Arg-17 Gly and B.beta.14 Arg-15 Gly, releasing two molecules of fibrinopeptide A and two molecules of fibrinopeptide B from the dimeric fibrinogen molecule and leaving behind a fibrin monomer capable of polymerization. The removal of fibrinopeptide A by thrombin proceeds at a much faster rate than that of fibrinopeptide B; however, the removal of fibrinopeptide A alone is sufficient for polymerization to occur.
It has been postulated that polymerization of fibrin monomer molecules takes place through two proceses, initially by an end-to-end association of molecules forming a fibrin strand and secondarily, by a lateral association of the strands. Light scattering has been used to differentiate between the two steps in polymerization. Recently the same technique was used to demonstrate that the removal of fibrinopeptide A led to the polymerization of fibrin monomers to form protofibrils and that the effect of the removal of fibrinopeptide B is an enhancement of the rate of lateral association of protofibrils.
Recent investigations have focused on defining areas of the fibrin monomer molecule that participtate in polymerization by identifying binding properties of degradation products of fibrinogen or fibrin. It has been shown that a certain abnormal fibrinogen has a functional binding site in the Fragment D domain but a defective site in the NH.sub.2 -terminal region. This fibrinogen variant contains a single amino acid substitution, A.alpha.19 Arg.fwdarw.Ser, suggesting that the NH.sub.2 -terminal binding site is at or near residue 19 on the A.alpha. chain. Recently, evidence was provided that the tripeptide Gly-Pro-Arg, which is contiguous with fibrinopeptide A, binds to fibrinogen and inhibits fibrin monomer polymerization. This suggests that an NH.sub.2 -terminal binding site is located on the A.alpha. chain of fibrinogen immediately following fibrinopeptide A.
It has been postulated that the Fragment D binding site is located on either the NH.sub.2 -terminal or COOH-terminal region of the .gamma. chain remnant of the Fragment D. However, the Fragment D binding site was not known prior to the present invention.
Although the binding-site was not known, several drugs that interfere with the formation of cross-linked fibrin were known, for example, heparin, the coumarins, and the indanedione drugs. The point of action and the mechanism of these drugs is not completely understood, and some undesirable side effects are known. For example, heparin has been known to occasionally cause severe thrombocytopenia (decrease in the number of platelets). The orally administered coumarins can cause nonspecific dermatitis and even skin necrosis. The indanediones (e.g. phenindione) have antithyroid activity and can cause liver and kidney damage. Accordingly, other anticoagulants, having a known and specific course of action and therefore being more easily controllable, are highly desirable.